Recent advances in the areas of medicine and biotechnology have led to the increased isolation and development of biologically active and therapeutically or diagnostically useful peptides and proteins. However, peptides and proteins generally have limited stability (half-life) physiologically, and are rapidly degraded. Additionally, peptides and proteins can have difficulty in efficiently interacting with or crossing cell membranes. Peptides and proteins are generally comprised of both charged and uncharged amino acid residues that impart structure and solubility to the peptide or protein. However, this charge can hinder membrane binding and membrane transport in cells. Therefore the development of methods for delivering peptides and proteins to cells is a current and continuing need in the areas of research and therapeutics.
Hydrophobic modification of compounds is known to increase binding of the compound to the cellular membranes. Recently, several examples of hydrophobic modification of peptides and proteins have been described in the literature (Storch et al. 1996; Kamyshny et al. 1997; Wang et al. 1997; Wang et al. 1999; Schreier et al. 2000; Wang et al. 2000; Wang et al. 2002; Wang et al. 2003). Hydrophobic modification entails the covalent attachment of one or more hydrophobic substituents to the polypeptide, usually through a nitrogen atom on the polypeptide. This modification can be accomplished through the use of chemically stable or chemically labile groups. If the attachment group is chemically labile, the bond can cleave at a certain rate under physiological conditions in order to allow the hydrophobic substituents to separate from the polypeptide. Following delivery of the hydrophobized polypeptide, several internalization pathways are possible. For example the complex can bind to the cellular membrane and either be endocytosed, directly transverse the membrane, or be trapped in or on the membrane. The complex can also be endocytosed and then cross internal membranes. In addition to the previously mentioned possibilities, if the hydrophobic group is labile, the hydrophobic group may cleave from the polypeptide in or near the cell. The polypeptide can then enter the cell via the mechanisms that are possible for the internalization of an unmodified polypeptide.
Several compound classes have been shown to be effective linkers for the hydrophobation, including simple acylation. Acylation with fatty acids or a variety of cyclic anhydrides are examples. Cyclic anhydrides have been shown a great deal of interest due to the ability of the linkage following the modification of the polypeptide. For example, maleic anhydrides have been previously utilized for reversible amine modification. The resulting maleamic acids are known to be stable under basic conditions, but hydrolyze rapidly under acidic conditions. Hydrophobic modification has even shown promise for the oral delivery of protein complexes. For example, Wang et al. has described the oral delivery of salmon calcitonin through the use of a reversible hydrophobation of the protein (Wang et al. 2003).
In addition to hydrophobation, several additional methods have been developed for the delivery of polypeptides to cells. Liposomal and micellar delivery, polymer conjugates, and systems involving combinations of polymers and lipids have all been used for the delivery of polypeptides to cells (Trubetskoy et al. 1993; Bijsterbosch et al. 1994; Rao et al. 1997; Yoshikawa et al. 1997; Capan et al. 1999; Schibli et al. 1999a; Schibli et al. 1999b; Montserret et al. 2000; Rao et al. 2000; Betz et al. 2001; Futaki et al. 2001; Gupta et al. 2001; Kisel et al. 2001; Nagy et al. 2001; Wang et al. 2001; Zelphati et al. 2001; Caliceti, et al. 2003; Copland et al. 2003; Mahato et al. 2003; Tiyaboonchai et al. 2003; Yang et al. 2003). Currently, there are several kits available from a variety of manufactures for the delivery of polypeptides to cell in vitro based on cationic lipids. However, the liposomal delivery of polypeptides has not been shown to be general. Since charge is the controlling factor in the binding of the peptide with the lipid, widely variable results would be expected based on the charge of the peptide. In the molecular conjugate area, several examples of increased serum half lives have been demonstrated following conjugations with a polymer. In all of these areas, there is a great deal of examples in which the lipid/liposome, micelle, and polymer conjugates are targeted to a cell utilizing some type of polypeptide acting as a targeting ligand.